Microorganisms and assays for the identification of antibiotics

ABSTRACT

The present invention features methods for the identification of compounds and compositions useful as antibiotics and antibacterial agents. In particular, the invention features methods for the identification of modulators of a previously unidentified target protein, termed CoaX. High-throughput assay systems are featured as well as assay kits for the identification of CoaX modulators. Also featured are coaX nucleic acid molecules and purified CoaX proteins, as well as recombinant vectors and microorganisms including the gene, coaX.

RELATED APPLICATIONS

This application is a divisional application of. U.S. patent applicationSer. No. 11/011,979, entitled “Microorganisms and Assays for theIdentification of Antibiotics”, filed Dec. 13, 2004, which is adivisional application of U.S. patent application Ser. No. 09/813453,entitled “Microorganisms and Assays for the Identification ofAntibiotics” filed Mar. 20, 2001, now U.S. Pat. No. 6,830,898B2, whichclaims the benefit of prior filed provisional U.S. Patent ApplicationSer. No. 60/227,860, entitled “Novel Microbial Pantothenate Kinase Geneand Methods of Use”, filed Aug. 24, 2000, which is also related to U.S.patent application Ser. No. 09/667,569, entitled “Methods andMicroorganisms for Production of Panto-Compounds”, filed Sep. 21, 2000(pending). The entire contents of the above-referenced patentapplications are incorporated herein by this reference.

BACKGROUND OF THE INVENTION

Antimicrobial or antibiotic treatment is a well-accepted therapy forfighting microbial infections that takes advantage of the existence ofbiological processes that are unique to bacteria or fungi that can besafely inhibited to the detriment of the bacteria, without producingundesired or harmful side effects in the individual receiving suchtherapy. However, due at least in part to the continual evolution ofmicrobial resistance to the available classes of antibiotics, and inpart to the recent slowdown in the introduction of novel antimicrobialsto market, there exists a need for the development of screening assaysthat target previously unexploited biochemical systems in microbes. Inparticular, there exists the need for the identification of newbacterial targets for use in drug discover programs designed to identifyagents having potential use as anti-infective agents with novel modes ofactions.

SUMMARY OF THE INVENTION

The present invention is based at least in part, on the identificationof a novel target for use in screening assays designed to identifyantimicrobial agents. In particular, the present invention is based onthe identification and characterization of a previously unidentifiedmicrobial pantothenate kinase gene, coaX. The coaX gene was firstidentified in B. subtilis where it is one of two genes encodingfunctional pantothenate kinase. Initially the present inventorsidentified and cloned the B. subtilis coaA gene (previously termed yqjs)that encodes a pantothenate kinase homologous to the CoaA enzymepreviously characterized in E. coli. A second gene (previously termedyacB) has also been identified and cloned by the present inventors thatis not homologous to any previously described pantothenate kinase. Thislatter pantothenate kinase-encoding gene has been renamed coaX. The coaXgene could be deleted from B. subtilis strains with an intact coaA gene,but it could not be deleted from a strain containing a deletion in thecoaA gene, indicating that the coaX gene is not essential in B. subtilisstrains with a wild-type coaA gene. Homologs of the coaX gene can befound in a number of bacterial species, including but not limited toAquifex aeolicus, Bacillus anthracis, Bacillus halodurans, Bacillusstearothermophilus, Caulobacter crescentus, Chlorobium tepidum,Clostridium acetobutylicum, Dehalococcoides ethenogenes, Deinococcusradiodurans, Desulfovibrio vulgaris, Geobacter sulfurreducens,Pseudomonas putida, Rhodobacter capsulatus, Thiobacillus ferrooxidans,Streptomyces coelicolor, Synechocystis sp., Thermotoga maritima,Bordetella pertussis, Borrelia burgdorferi, Campylobacter jejuni,Clostridium difficile, Helicobacter pylori, Neisseria meningitidis,Neisseria gonorrhoeae, Porphyromonas gingivalis, Pseudomonas aeruginosa,Pseudomonas syringae pv tomato, Treponema pallidum, Xylella fastidiosaand Mycobacterium tuberculosis. More importantly, however, this novelpantothenate kinase gene has been found to be the sole essentialpantothenate kinase in troublesome pathogens including, but not limitedto, Bordetella pertussis, Borrelia burgdorferi, Campylobacter jejuni,Helicobacter pylori, Neisseria meningitidis, Pseudomonas aeruginosa,Treponema pallidum and Xylella fastidiosa. Accordingly, the coaX generepresents an attractive target for screening for new antibacterialcompounds to combat these pathogenic microorganisms, particularlymicroorganisms in which coaX is the sole pantothenate kinase-encodinggene.

Accordingly, the present invention features isolated CoaX proteins, inparticular, proteins encoded by the coaX gene in bacteria. The inventionalso features isolated nucleic acid molecules and/or genes, e.g.,bacterial nucleic acid molecules and/or genes, in particular, isolatedbacterial coaX nucleic acid molecules and/or genes. Also featured arevectors that contain isolated coaX nucleic acid molecules and/or genesas well as mutant coaX nucleic acid molecules and/or genes. Alsofeatured are recombinant microorganisms (e.g., microorganisms belongingto the genus Escherichia or Bacillus, for example, E. coli or B.subtilis) containing isolated coaX nucleic acid molecules and/or genesor mutant coaX nucleic acid molecules and/or genes of the presentinvention. In particular, the invention features recombinantmicroorganisms that produce the CoaX proteins of the present invention,e.g., pantohthenate kinase proteins encodes by the coaX nucleic acidmolecules and/or genes of the present invention.

Also featured are methods for identifying CoaX modulators utilizing, forexample, isolated CoaX proteins of the present invention or recombinantmicroorganisms expressing the CoaX proteins of the present invention.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the Coenzyme A biosyntheticpathway in E. coli.

FIG. 2 is a schematic representation of the structure of the Bacillussubtilis genome in the region of the coaA gene. The scale is in basepairs and the significant open reading frames are shown by open arrows.

FIG. 3 is a schematic representation of the structure of pAN296, aplasmid designed to delete most of the B. subtilis coaA gene andsubstitute a chloramphenicol resistance gene.

FIG. 4 is a schematic representation of the structure of the Bacillussubtilis genome in the region of the coaX (yacB) gene. The scale is inbase pairs, the significant open reading frames are shown by open arrowsand certain predicted restriction fragments are indicated by thick bars.

FIG. 5 is a schematic representation of the structure of pAN341 andpAN342, two independent PCR-derived clones of B. subtilis yacB (renamedherein as coaX).

FIG. 6A-D depicts a multiple sequence alignment (MSA) of the amino acidsequences encoded by fourteen known or predicted microbial coaX genes.SEQ ID NOs:2-15 correspond to the amino acid sequences of Bacillussubtilis (SwissProt™ Accession No. P37564), Clostridium acetobulyticum(WIT™ Accession No. RCA03301, Argonne National Laboratories),Streptomyces coelicolor (PIR™ Accession No. T36391), Mycobacteriumtuberculosis (SwissProt™ Accession No. 006282), Rhodobacter capsulatus(WIT™ Accession No. RRC02473), Desulfovibrio vulgaris (DBJ™ AccessionNo. BAA21476.1), Deinococcus radiodurans (SwissProt™ Accession No.Q9RX54), Thermotoga maritima (GenBank™ Accession No. AAD35964.1),Treponema pallidum (SwissProt™ Accession No. 083446), Borreliaburgdorferi (SwissProt™ Accession No. 051477), Aquifex aeolicus(SwissProt™ Accession No. 067753), Synechocystis sp. (SwissProt™Accession No. P74045), Helicobacter pylori (SwissProt™ Accession No.025533), and Bordetella pertussis (SwissProt™ Accession No. Q45338),respectively. The alignment was generated using ClustalW MSA software atthe GenomeNet CLUSTALW Server at the Institute for Chemical Research,Kyoto University. The following parameters were used: PairwiseAlignment, K-tuple (word) size=1, Window size=5, Gap Penalty=3, Numberof Top Diagonals=5, Scoring Method=Percent; Multiple Alignment, Gap OpenPenalty=10, Gap Extension Penalty=0.0, Weight Transition=No, Hydrophilicresidues=Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg and Lys, HydrophobicGaps=Yes; and Scoring Matrix=BLOSUM.

FIG. 7 is a schematic representation of the structure of pAN336, aplasmid designed to delete B. subtilis coaX from its chromosomal locusand replace it with a kanamycin resistence gene.

FIG. 8 is a schematic representation of the construction of pOTP72, aplasmid containing the H. pylori coaX gene.

FIG. 9 is a schematic representation of the construction of pOTP73, aplasmid containing the P. aeruginosa coaX gene.

FIG. 10 is a schematic representation of the construction of pOTP71, aplasmid containing the B. subtilis coaX gene.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based at least in part, on the identificationof a novel target for use in screening assays designed to identifyantimicrobial agents. In particular, the present invention is based onthe identification and characterization of a previously unidentifiedmicrobial pantothenate kinase. This pantothenate kinase, encoded by agene, termed coaX herein, is structurally unrelated to the previouslycharacterized E. coli pantothenate gene, coaA, however, both genesencode functional pantothenate kinase enzymes, pantothenate kinase beingessential for the synthesis of Coenzyme A (CoA). CoA is an essentialcoenzyme in all cells, participating in over 100 different intermediaryreactions in cellular metabolism including, but not limited to, thetricarboxylic acid (TCA) cycle, fatty acid metabolism, vitaminbiosynthesis and numerous other reactions of intermediary metabolism.Accordingly, pantothenate kinase production is essential for microbialgrowth. Coenzyme A (CoA) is synthesized in both eukaryotes andprokaryotes from pantothenate, also known as pantothenic acid or vitaminB5. The initial (and possibly rate-controlling) step in the conversionof pantothenate to Coenzyme A (CoA) is phosphorylation of pantothenateby pantothenate kinase. A schematic representation of the pathwayleading to CoaA biosynthesis in E. coli, i.e., the E. coli CoAbiosynthetic pathway is set forth as FIG. 1. The term “CoA biosyntheticpathway”, as used herein, includes the biosynthetic pathway involvingCoA biosynthetic enzymes (e.g., polypeptides encoded by biosyntheticenzyme-encoding genes), compounds (e.g., precursors, substrates,intermediates or products), cofactors and the like utilized in theformation or synthesis of CoA from pantothenate. The CoA biosyntheticpathway depicted is also presumed to be that utilized by othermicroorganisms. The term “CoA biosynthetic pathway” includes thebiosynthetic pathway leading to the synthesis of CoA in microorganisms(e.g., in vivo) as well as the biosynthetic pathway leading to thesynthesis of CoA in vitro.

The term “Coenzyme A or CoA biosynthetic enzyme” includes any enzymeutilized in the formation of a compound (e.g., intermediate or product)of the CoA biosynthetic pathway, for example, the coaA, panK or coaXgene product which catalyzes the phosphorylation of pantothenate to form4′-phosphopantothenate, or the coaD gene product which catalyzes theconversion of 4′-phosphopantetheine to dephosphocoenzyme A.

The coaX gene was first identified in B. subtilis, a microorganism inwhich it is one of two pantothenate kinase-encoding genes. Initially,the present inventors identified and cloned the B. subtilis coaA gene(previously termed yqjs) that encodes a pantothenate kinase homologousto the CoaA enzyme previously characterized in E. coli. A second gene(previously termed yacB) has also been identified and cloned by thepresent inventors that is not homologous to any previously describedpantothenate kinase. This latter pantothenate kinase-encoding gene hasbeen renamed coaX. The coaX gene could be deleted from B. subtilisstrains with an intact coaA gene, but it could not be deleted from astrain containing a deletion in the coaA gene, indicating that the coaXgene is not essential in B. subtilis strains with a wild-type coaA gene.

Homologs of the coaX gene can be found in a number of bacterial species,including but not limited to Aquifex aeolicus, Bacillus anthracis,Bacillus halodurans, Bacillus stearothermophilus, Caulobactercrescentus, Chlorobium tepidum, Clostridium acetobutylicum,Dehalococcoides ethenogenes, Deinococcus radiodurans, Desulfovibriovulgaris, Geobacter sulfurreducens, Pseudomonas putida, Rhodobactercapsulatus, Thiobacillus ferrooxidans, Streptomyces coelicolor,Synechocystis sp., Thermotoga maritima, Bordetella pertussis, Borreliaburgdorferi, Campylobacter jejuni, Clostridium difficile, Helicobacterpylori, Neisseria meningitidis, Neisseria gonorrhoeae, Porphyromonasgingivalis, Pseudomonas aeruginosa, Legionella pneumophila, Treponemapallidum, Xylella fastidiosa and Mycobacterium tuberculosis. Moreimportantly, however, this novel pantothenate kinase gene has been foundto be the sole essential pantothenate kinase in troublesome pathogensincluding, but not limited to, Bordetella pertussis, Borreliaburgdorferi, Campylobacter jejuni, Helicobacter pylori, Neisseriameningitidis, Pseudomonas aeruginosa, Treponema pallidum and Xylellafastidiosa. Accordingly, the coaX gene represents an attractive targetfor screening for new antibacterial compounds to combat these pathogenicmicroorganisms, particularly microorganisms in which coaX is the solepantothenate kinase-encoding gene.

Accordingly, in one aspect the present invention features assays for theidentification an antibiotic that involve contacting a compositioncomprising a CoaX protein with a test compound; and determining theability of the test compound to inhibit the activity of the CoaXprotein; wherein the compound is identified as an antibiotic based onthe ability of the compound to inhibit the activity of the CoaX protein.In another aspect, the invention features an assay for theidentification a potential antibiotic that involves contacting an assaycomposition comprising CoaX with a test compound; and determining theability of the test compound to bind to the CoaX; wherein the compoundis identified as a potential antibiotic based on the ability of thecompound to bind to the CoaX. In a preferred assay format, thecomposition is also contacted with pantothenate or a pantothenate analogand activity determined.

In another aspect, the invention features methods for identifyingpantothenate kinase modulators that involve contacting a recombinantcell expressing a single pantothenate kinase encoded by a coaX gene witha test compound and determining the ability of the test compound tomodulate pantothenate kinase activity in said cell. In another aspect,the invention features methods for identifying pantothenate kinasemodulators that involve contacting a recombinant cell expressing a firstand second pantothenate kinase, with a test compound and determining theability of the test compound to modulate pantothenate kinase activity insaid cell, wherein the first or second pantothenate kinase has reducedactivity. Preferred recombinant microorganisms are of the genus Bacillusor Escherichia (e.g., Bacillus subtilis or Escherchia coli).

Also featured are isolated nucleic acid molecules that include a coaXgene of the present invention, isolated proteins encoded by the coaXgenes of the present invention and biologically active portions thereof.In one embodiment, the invention features a coaX gene derived from amicroorganism selected from the group consisting of Aquifex aeolicus,Bacillus anthracis, Bacillus halodurans, Bacillus stearothermophilus,Bacillus subtilis, Caulobacter crescentus, Chlorobium tepidum,Clostridium acetobutylicum, Dehalococcoides ethenogenes, Deinococcusradiodurans, Desulfovibrio vulgaris, Geobacter sulfurreducens,Pseudomonas putida, Rhodobacter capsulatus, Thiobacillus ferrooxidans,Streptomyces coelicolor, Synechocystis sp., Thermotoga maritima,Bordetella pertussis, Borrelia burgdorferi, Campylobacter jejuni,Clostridium difficile, Helicobacter pylori, Neisseria meningitidis,Neisseria gonorrhoeae, Porphyromonas gingivalis, Pseudomonas aeruginosa,Legionella pneumophila, Treponema pallidum, Xylella fastidiosa andMycobacterium tuberculosis, or a protein encoded by said coaX gene.

In another embodiment, the invention features isolated nucleic acidmolecules that include a coaX gene derived from a pathogenic bacteriumselected from the group consisting of Bacillus anthracis, Bordetellapertussis, Borrelia burgdorferi, Campylobacter jejuni, Clostridiumdifficile, Helicobacter pylori, Neisseria meningitidis, Neisseriagonorrhoeae, Pseudomonas aeruginosa, Porphyromonas gingivalis,Legionella pneumophila, Treponema pallidum and Xylella fastidiosa, or aprotein encoded by said coaX gene. In a preferred embodiment, theinvention features isolated nucleic acid molecules that include a coaXgene derived from a pathogenic bacterium selected from the groupconsisting of Bordetella pertussis, Borrelia burgdorferi, Campylobacterjejuni, Helicobacter pylori, Neisseria meningitidis, Pseudomonasaeruginosa, Treponema pallidum and Xylella fastidiosa, or a proteinencoded by said coaX gene.

Also featured are recombinant vectors that include the isolated coaXgenes of the present invention and recombinant microorganisms thatinclude said vectors.

I. General Background

A pantothenate kinase activity was first identified in Salmonellatyphimurium by screening for temperature-sensitive mutants whichsynthesized CoA at permissive temperatures but excreted pantothenate atnon-permissive temperatures. The mutations were mapped in the Salmonellachromosome and the genetic locus was designated coaA. The gene encodesthe enzyme that catalyzes the first step in the biosynthesis of coenzymeA from pantothenate (Dunn and Snell (1979) J. Bacteriol. 140:805-808).Escherichia coli temperature sensitive mutants have also been isolatedand characterized (Vallari and Rock (1987) J. Bacteriol. 169:5795-5800).These mutants (named coaA15(Ts)) are defective in the conversion ofpantothenate to CoA and further exhibit a temperature-sensitive growthphenotype, indicating that pantothenate kinase activity is essential forgrowth. Moreover, it was noted that CoA inhibited pantothenate kinaseactivity to the same degree in the mutant as compared to the wild-typeenzyme.

Feedback resistant E. coli mutants (named coaA16(Fr)) have also beenisolated that possess a pantothenate kinase activity that is refractoryto feedback inhibition by CoA (Vallari and Jackowski (1988) J.Bacteriol. 170:3961-3966). The mutation responsible for the reversionis, suprisingly, not genetically linked to the coaA gene bytransduction. Additional data described therein support the view thatthe total cellular CoA content is controlled by both modulation ofbiosynthesis at the pantothenate kinase step and possibly by degradationof CoA to 4′-phosphopantetheine.

The wild-type E. coli coaA gene was cloned by functional complementationof E. coli temperature-sensitive mutants. The sequence of the wild-typegene was determined (Song and Jackowski (1992) J. Bacteriol.174:6411-6417 and Flamm et al. (1988) Gene (Amst.) 74:555-558). Strainscontaining multiple copies of the coaA gene possessed 76-fold higherspecific activity of pantothenate kinase, however, there was only a2.7-fold increase in the steady state level of CoA (Song and Jackowski,supra). It has further been reported that the prokaryotic enzyme(encoded by coaA in E. coli and a variety of other microorganisms) isfeedback inhibited by CoA both in vivo and in vitro with CoA being aboutfive times more potent than acetyl-CoA in inhibiting the enzyme (Songand Jackowski, supra and Vallari et al., supra). These data furthersupport the view that feedback inhibition of pantothenate kinaseactivity is a critical factor controlling intracellular CoAconcentration. The E. coli CoaA protein has been crystalized and thestructure solved (Yun et al. (2000) J. Biol. Chem. 275(36):28093-28099).

Using standard search and alignment tools, coaA homologues have beenidentified in Hemophilus influenzae, Mycobacterium tuberculosis, Vibriocholerae, Streptococcus pyogenes and Bacillus subtilis. By contrast,proteins with significant similarity could not be identified ineukaryotic cells including Saccharomyces cerevisiae or in mammalianexpressed sequence tag (EST) databases. Using a genetic selectionstrategy, a cDNA encoding pantothenate kinase activity has recently beenidentified from Aspergillus nidulans (Calder et al. (1999) J. Biol.Chem. 274:2014-2020). The eukaryotic pantothenate kinase gene (panK) hasdistinct primary structure and unique regulatory properties that clearlydistinguish it from its prokaryotic counterpart. A mammalianpantothenate kinase gene (mpanK1α) has also been isolated which encodesa protein having homology to the A. nidulans PanK protein and to thepredicted gene product of GenBank™ Accession Number 927798 identified inthe S. cerevisiae genome (Rock et al. (2000) J. Biol. Chem.275:1377-1383).

II. CoaX Nucleic Acid Molecules

The present invention relates, at least in part, to the identificationof a novel microbial pantothenate kinase encoding gene, coaX, that isstructurally distinct from a previously identified microbialpantothenate kinase encoding gene, coaA. Accordingly, one aspect of thepresent invention features isolated coaX nucleic acid molecules and/orgenes useful, for example, for encoding pantothenate kinase enzymes foruse in screening assays.

The term “nucleic acid molecule” includes DNA molecules (e.g., linear,circular, cDNA or chromosomal DNA) and RNA molecules (e.g., tRNA, rRNA,mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. The term “isolated” nucleic acidmolecule includes a nucleic acid molecule that is free of sequences thatnaturally flank the nucleic acid molecule (i.e., sequences located atthe 5′ and 3′ ends of the nucleic acid molecule) in the chromosomal DNAof the organism from which the nucleic acid is derived. In variousembodiments, an isolated nucleic acid molecule can contain less thanabout 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, 0.1 kb, 50 bp, 25 bpor 10 bp of nucleotide sequences which naturally flank the nucleic acidmolecule in chromosomal DNA of the microorganism from which the nucleicacid molecule is derived. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterials when produced by recombinant techniques, or substantially freeof chemical precursors or other chemicals when chemically synthesized.

The term “gene”, as used herein, includes a nucleic acid molecule (e.g.,a DNA molecule or segment thereof), for example, a protein orRNA-encoding nucleic acid molecule, that in an organism, is separatedfrom another gene or other genes, by intergenic DNA (i.e., interveningor spacer DNA which naturally flanks the gene and/or separates genes inthe chromosomal DNA of the organism). A gene may direct synthesis of anenzyme or other protein molecule (e.g., may comprise coding sequences,for example, a contiguous open reading frame (ORF) which encodes aprotein) or may itself be functional in the organism. A gene in anorganism, may be clustered in an operon, as defined herein, said operonbeing separated from other genes and/or operons by the intergenic DNA.Individual genes contained within an operon may overlap withoutintergenic DNA between said individual genes. An “isolated gene”, asused herein, includes a gene which is essentially free of sequenceswhich naturally flank the gene in the chromosomal DNA of the organismfrom which the gene is derived (i.e., is free of adjacent codingsequences which encode a second or distinct protein or RNA molecule,adjacent structural sequences or the like) and optionally includes 5′and 3′ regulatory sequences, for example promoter sequences and/orterminator sequences. In one embodiment, an isolated gene includespredominantly coding sequences for a protein (e.g., sequences whichencode Bacillus proteins). In another embodiment, an isolated geneincludes coding sequences for a protein (e.g., for a Bacillus protein)and adjacent 5′ and/or 3′ regulatory sequences from the chromosomal DNAof the organism from which the gene is derived (e.g., adjacent 5′ and/or3′ Bacillus regulatory sequences). Preferably, an isolated gene containsless than about 10 kb, 5 kb, 2 kb, 1 kb, 0.5 kb, 0.2 kb, 0.1 kb, 50 bp,25 bp or 10 bp of nucleotide sequences which naturally flank the gene inthe chromosomal DNA of the organism from which the gene is derived.

In one embodiment, an isolated nucleic acid molecule is or includes acoaX gene. In another embodiment, an isolated nucleic acid molecule isor includes a portion or fragment of a coaX gene. In one embodiment, anisolated coaX nucleic acid molecule is derived from a microorganismselected form the group consisting of Aquifex aeolicus, Bacillusanthracis, Bacillus halodurans, Bacillus stearothermophilus, Bacillussubtilis, Caulobacter crescentus, Chlorobium tepidum, Clostridiumacetobutylicum, Dehalococcoides ethenogenes, Deinococcus radiodurans,Desulfovibrio vulgaris, Geobacter sulfurreducens, Pseudomonas putida,Rhodobacter capsulatus, Thiobacillus ferrooxidans, Streptomycescoelicolor, Synechocystis sp., Thermotoga maritima, Bordetellapertussis, Borrelia burgdorferi, Campylobacter jejuni, Clostridiumdifficile, Helicobacter pylori, Neisseria meningitidis, Neisseriagonorrhoeae, Porphyromonas gingivalis, Pseudomonas aeruginosa,Pseudomonas syringae pv tomato, Treponema pallidum, Xylella fastidiosa,Legionella pneumophila and Mycobacterium tuberculosis. In anotherembodiment, an isolated coaX nucleic acid molecule is derived from amicroorganism selected from the group consisting of Bacillus anthracis,Bordetella pertussis, Borrelia burgdorferi, Campylobacter jejuni,Clostridium difficile, Helicobacter pylori, Neisseria meningitidis,Neisseria gonorrhoeae, Porphyromonas gingivalis, Pseudomonas aeruginosa,Treponema pallidum, Xylella fastidiosa and Legionella pneumophila. Inanother embodiment, an isolated coaX nucleic acid molecule is derivedfrom a microorganism selected from the group consisting of Bordetellapertussis, Borrelia burgdorferi, Campylobacter jejuni, Clostridiumdifficile, Helicobacter pylori, Neisseria meningitidis, Pseudomonasaeruginosa, Treponema pallidum and Xylella fastidiosa. In anotherembodiment, an isolated coaX nucleic acid molecule or gene comprises anucleotide sequence set forth as any one of SEQ ID NOs:SEQ ID NO:32, SEQID NO:69, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:1, SEQ IDNO:38, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:23, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:28, SEQ ID NO:60, SEQ IDNO:27, SEQ ID NO:34 or SEQ ID NO:68, SEQ ID NO:25, SEQ ID NO:40, SEQ IDNO:44, SEQ ID NO:42, SEQ ID NO:35 or SEQ ID NO:37, SEQ ID NO:62, SEQ IDNO:26, SEQ ID NO:24, SEQ ID NO:33, SEQ ID NO:29, SEQ ID NO:64, SEQ IDNO:30 and SEQ ID NO:66. In another embodiment, an isolated nucleic acidmolecule of the present invention comprises a nucleotide sequence whichis at least about 50-55%, preferably at least about 60-65%, morepreferably at least about 70-75%, more preferably at least about 80-85%,and even more preferably at least about 90-95% or more identical to anucleotide sequence set forth as any one of SEQ ID NOs:SEQ ID NO:32, SEQID NO:69, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:1, SEQ IDNO:38, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:23, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:28, SEQ ID NO:60, SEQ IDNO:27, SEQ ID NO:34 or SEQ ID NO:68, SEQ ID NO:25, SEQ ID NO:40, SEQ IDNO:44, SEQ ID NO:42, SEQ ID NO:35 or SEQ ID NO:37, SEQ ID NO:62, SEQ IDNO:26, SEQ ID NO:24, SEQ ID NO:33, SEQ ID NO:29, SEQ ID NO:64, SEQ IDNO:30 and SEQ ID NO:66.

In yet another embodiment, an isolated coaX nucleic acid molecule orgene comprises a nucleotide sequence that encodes a protein having anamino acid sequence as set forth in any one of SEQ ID NOs:SEQ ID NO:12,SEQ ID NO:70, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:2, SEQID NO:51, SEQ ID NO:53, SEQ ID NO:3, SEQ ID NO:57, SEQ ID NO:8, SEQ IDNO:59, SEQ ID NO:7, SEQ ID NO:61, SEQ ID NO:6, SEQ ID NO:63, SEQ IDNO:4, SEQ ID NO:13, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:11, SEQ IDNO:21, SEQ ID NO:55, SEQ ID NO:14 or SEQ ID NO:67, SEQ ID NO:43 or SEQID NO:22, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:20, SEQ ID NO:10, SEQ IDNO:65 and SEQ ID NO:5. In yet another embodiment, an isolated coaXnucleic acid molecule or gene encodes a homologue of the CoaX proteinshaving the amino acid sequences of SEQ ID NOs:SEQ ID NO:12, SEQ IDNO:70, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:2, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:3, SEQ ID NO:57, SEQ ID NO:8, SEQ IDNO:59, SEQ ID NO:7, SEQ ID NO:61, SEQ ID NO:6, SEQ ID NO:63, SEQ IDNO:4, SEQ ID NO:13, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:11, SEQ IDNO:21, SEQ ID NO:55, SEQ ID NO:14 or SEQ ID NO:67, SEQ ID NO:43 or SEQID NO:22, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:20, SEQ ID NO:10, SEQ IDNO:65 and SEQ ID NO:5. As used herein, the term “homologue” includes aprotein or polypeptide sharing at least about 30-35%, preferably atleast about 35-40%, more preferably at least about 40-50%, and even morepreferably at least about 60%, 70%, 80%, 90% or more identity with theamino acid sequence of a wild-type protein or polypeptide describedherein and having a substantially equivalent functional or biologicalactivity as said wild-type protein or polypeptide. For example, a CoaXhomologue shares at least about 30-35%, preferably at least about35-40%, more preferably at least about 40-50%, and even more preferablyat least about 60%, 70%, 80%, 90% or more identity with any one of theproteins having the amino acid sequences set forth as SEQ ID NOs:SEQ IDNO:12, SEQ ID NO:70, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:2, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:3, SEQ ID NO:57, SEQ IDNO:8, SEQ ID NO:59, SEQ ID NO:7, SEQ ID NO:61, SEQ ID NO:6, SEQ IDNO:63, SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:9, SEQ ID NO:15, SEQ IDNO:11, SEQ ID NO:21, SEQ ID NO:55, SEQ ID NO:14 or SEQ ID NO:67, SEQ IDNO:43 or SEQ ID NO:22, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:20, SEQ IDNO:10, SEQ ID NO:65 and SEQ ID NO:5 and has a substantially equivalentfunctional or biological activity (i.e., is a functional equivalent) ofthe proteins having the amino acid sequences set forth as SEQ ID NOs:SEQID NO:12, SEQ ID NO:70, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:2, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:3, SEQ ID NO:57, SEQ IDNO:8, SEQ ID NO:59, SEQ ID NO:7, SEQ ID NO:61, SEQ ID NO:6, SEQ IDNO:63, SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:9, SEQ ID NO:15, SEQ IDNO:11, SEQ ID NO:21, SEQ ID NO:55, SEQ ID NO:14 or SEQ ID NO:67, SEQ IDNO:43 or SEQ ID NO:22, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:20, SEQ IDNO:10, SEQ ID NO:65 and SEQ ID NO:5 (e.g., has a substantiallyequivalent CoaX activity). In a preferred embodiment, an isolated coaXnucleic acid molecule or gene comprises a nucleotide sequence thatencodes a polypeptide as set forth in any one of SEQ ID NOs:SEQ IDNO:12, SEQ ID NO:70, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:2, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:3, SEQ ID NO:57, SEQ IDNO:8, SEQ ID NO:59, SEQ ID NO:7, SEQ ID NO:61, SEQ ID NO:6, SEQ IDNO:63, SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:9, SEQ ID NO:15, SEQ IDNO:11, SEQ ID NO:21, SEQ ID NO:55, SEQ ID NO:14 or SEQ ID NO:67, SEQ IDNO:43 or SEQ ID NO:22, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:20, SEQ IDNO:10, SEQ ID NO:65 and SEQ ID NO:5.

In another embodiment, an isolated coaX nucleic acid molecule hybridizesto all or a portion of a nucleic acid molecule having the nucleotidesequence set forth in any one of SEQ ID NOs:SEQ ID NO:32, SEQ ID NO:69,SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:1, SEQ ID NO:38, SEQID NO:31, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:23, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:28, SEQ ID NO:60, SEQ ID NO:27, SEQ IDNO:34 or SEQ ID NO:68, SEQ ID NO:25, SEQ ID NO:40, SEQ ID NO:44, SEQ IDNO:42, SEQ ID NO:35 or SEQ ID NO:37, SEQ ID NO:62, SEQ ID NO:26, SEQ IDNO:24, SEQ ID NO:33, SEQ ID NO:29, SEQ ID NO:64, SEQ ID NO:30 and SEQ IDNO:66 or hybridizes to all or a portion of a nucleic acid moleculehaving a nucleotide sequence that encodes a polypeptide having the aminoacid sequence of any of SEQ ID NOs:SEQ ID NO:12, SEQ ID NO:70, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:2, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:3, SEQ ID NO:57, SEQ ID NO:8, SEQ ID NO:59, SEQ IDNO:7, SEQ ID NO:61, SEQ ID NO:6, SEQ ID NO:63, SEQ ID NO:4, SEQ IDNO:13, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:11, SEQ ID NO:21, SEQ IDNO:55, SEQ ID NO:14 or SEQ ID NO:67, SEQ ID NO:43 or SEQ ID NO:22, SEQID NO:39, SEQ ID NO:41, SEQ ID NO:20, SEQ ID NO:10, SEQ ID NO:65 and SEQID NO:5. Such hybridization conditions are known to those skilled in theart and can be found in Current Protocols in Molecular Biology, Ausubelet al., eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6.Additional stringent conditions can be found in Molecular Cloning: ALaboratory Manual, Sambrook et al., Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989), chapters 7, 9 and 11. A preferred,non-limiting example of stringent hybridization conditions includeshybridization in 4× sodium chloride/sodium citrate (SSC), at about65-70° C. (or hybridization in 4×SSC plus 50% formamide at about 42-50°C.) followed by one or more washes in 1×SSC, at about 65-70° C. Apreferred, non-limiting example of highly stringent hybridizationconditions includes hybridization in 1×SSC, at about 65-70° C. (orhybridization in 1×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 0.3×SSC, at about 65-70° C. A preferred,non-limiting example of reduced stringency hybridization conditionsincludes hybridization in 4×SSC, at about 50-60° C. (or alternativelyhybridization in 6×SSC plus 50% formamide at about 40-45° C.) followedby one or more washes in 2×SSC, at about 50-60° C. Ranges intermediateto the above-recited. values, e.g., at 65-70° C. or at 42-50° C. arealso intended to be encompassed by the present invention. SSPE (1×SSPEis 0.15 M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can besubstituted for SSC (1×SSC is 0.15 M NaCl and 15 mM sodium citrate) inthe hybridization and wash buffers; washes are performed for 15 minuteseach after hybridization is complete. The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be5-10° C. less than the melting temperature (T_(m)) of the hybrid, whereT_(m) is determined according to the following equations. For hybridsless than 18 base pairs in length, T_(m)(° C.)=2(# of A+T bases)+4(# ofG+C bases). For hybrids between 18 and 49 base pairs in length, T_(m)(°C.)=81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional preferred,non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C., see e.g., Churchand Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995, (or,alternatively, 0.2×SSC, 1% SDS). In another preferred embodiment, anisolated nucleic acid molecule comprises a nucleotide sequence that iscomplementary to a coaX nucleotide sequence as set forth herein (e.g.,is the full complement of the nucleotide sequence set forth as SEQ IDNO:19). Preferably, an isolated nucleic acid molecule of the inventionthat hybridizes under stringent conditions to the sequence of SEQ IDNO:SEQ ID NO:32, SEQ ID NO:69, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50,SEQ ID NO:1, SEQ ID NO:38, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:52, SEQID NO:54, SEQ ID NO:23, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:28, SEQ IDNO:60, SEQ ID NO:27, SEQ ID NO:34 or SEQ ID NO:68, SEQ ID NO:25, SEQ IDNO:40, SEQ ID NO:44, SEQ ID NO:42, SEQ ID NO:35 or SEQ ID NO:37, SEQ IDNO:62, SEQ ID NO:26, SEQ ID NO:24, SEQ ID NO:33, SEQ ID NO:29, SEQ IDNO:64, SEQ ID NO:30 and SEQ ID NO:66, or to a complement thereof,corresponds to a naturally-occurring nucleic acid molecule. As usedherein, a “naturally-occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature.

A nucleic acid molecule of the present invention (e.g., a coaX nucleicacid molecule or gene), can be isolated using standard molecular biologytechniques and the sequence information provided herein. For example,nucleic acid molecules can be isolated using standard hybridization andcloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F.,and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) or can be isolated by the polymerase chainreaction using synthetic oligonucleotide primers designed based upon thecoaX nucleotide sequences set forth herein, or flanking sequencesthereof. A nucleic acid of the invention (e.g., a coaX nucleic acidmolecule or gene), can be amplified using cDNA, mRNA or alternatively,chromosomal DNA, as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. Assays foridentifying coaX gene of the present invention or homologues thereof canbe accomplished, for example, by expressing the coaX gene in amicroorganism, for example, a microorganism which expresses pantothenatekinase in a temperature-sensitive manner, and assaying the gene for theability to complement a temperature sensitive (Ts) mutant forpantothenate kinase activity. A coaX gene that encodes a functionalpantothenate kinase is one that complements the Ts mutant.

Yet another embodiment of the present invention features mutant coaX andcoaA nucleic acid molecules or genes. The phrase “mutant nucleic acidmolecule” or “mutant gene” as used herein, includes a nucleic acidmolecule or gene having a nucleotide sequence which includes at leastone alteration (e.g., substitution, insertion, deletion) such that thepolypeptide or protein that may be encoded by said mutant exhibits anactivity that differs from the polypeptide or protein encoded by thewild-type nucleic acid molecule or gene. Preferably, a mutant nucleicacid molecule or mutant gene (e.g., a mutant coaA or coaX gene) encodesa polypeptide or protein having a reduced activity (e.g., having areduced pantothenate kinase activity) as compared to the polypeptide orprotein encoded by the wild-type nucleic acid molecule or gene, forexample, when assayed under similar conditions (e.g., assayed inmicroorganisms cultured at the same temperature). A mutant gene also canencode no polypeptide or have a reduced level of production of thewild-type polypeptide.

As used herein, a “reduced activity” or “reduced enzymatic activity” isone that is at least 5% less than that of the polypeptide or proteinencoded by the wild-type nucleic acid molecule or gene, preferably atleast 5-10% less, more preferably at least 10-25% less and even morepreferably at least 25-50%, 50-75% or 75-100% less than that of thepolypeptide or protein encoded by the wild-type nucleic acid molecule orgene. Ranges intermediate to the above-recited values, e.g., 75-85%,85-90%, 90-95%, are also intended to be encompassed by the presentinvention. As used herein, a “reduced activity” or “reduced enzymaticactivity” also includes an activity that has been deleted or “knockedout” (e.g., approximately 100% less activity than that of thepolypeptide or protein encoded by the wild-type nucleic acid molecule orgene). Activity can be determined according to any well accepted assayfor measuring activity of a particular protein of interest. Activity canbe measured or assayed directly, for example, measuring an activity of aprotein isolated or purified from a cell. Alternatively, an activity canbe measured or assayed within a cell or in an extracellular medium or ina crude extract of cells.

It will be appreciated by the skilled artisan that even a singlesubstitution in a nucleic acid or gene sequence (e.g., a basesubstitution that encodes an amino acid change in the correspondingamino acid sequence) can dramatically affect the activity of an encodedpolypeptide or protein as compared to the corresponding wild-typepolypeptide or protein. A mutant nucleic acid or mutant gene (e.g.,encoding a mutant polypeptide or protein), as defined herein, is readilydistinguishable from a nucleic acid or gene encoding a proteinhomologue, as described above, in that a mutant nucleic acid or mutantgene encodes a protein or polypeptide having an altered activity,optionally observable as a different or distinct phenotype in amicroorganism expressing said mutant gene or nucleic acid or producingsaid mutant protein or polypeptide (i.e., a mutant microorganism) ascompared to a corresponding microorganism expressing the wild-type geneor nucleic acid or producing said mutant protein or polypeptide. Bycontrast, a protein homologue has an identical or substantially similaractivity, optionally phenotypically indiscernable when produced in amicroorganism, as compared to a corresponding microorganism expressingthe wild-type gene or nucleic acid. Accordingly it is not, for example,the degree of sequence identity between nucleic acid molecules, genes,protein or polypeptides that serves to distinguish between homologuesand mutants, rather it is the activity of the encoded protein orpolypeptide that distinguishes between homologues and mutants:homologues having, for example, low (e.g., 30-50% sequence identity)sequence identity yet having substantially equivalent functionalactivities, and mutants, for example sharing 99% sequence identity yethaving dramatically different or altered functional activities.Exemplary homologues are set forth as SEQ ID NOs:SEQ ID NO:12, SEQ IDNO:70, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:3, SEQ ID NO:57, SEQ ID NO:8, SEQ ID NO:59, SEQ IDNO:7, SEQ ID NO:61, SEQ ID NO:6, SEQ ID NO:63, SEQ ID NO:4, SEQ IDNO:13, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:11, SEQ ID NO:21, SEQ IDNO:55, SEQ ID NO:14 or SEQ ID NO:67, SEQ ID NO:43 or SEQ ID NO:22, SEQID NO:39, SEQ ID NO:41, SEQ ID NO:20, SEQ ID NO:10, SEQ ID NO:65 and SEQID NO:5 (i.e., CoaX homologues). Exemplary mutants are described inExamples III-IV herein.

III. CoaX Proteins

Another aspect of the present invention features isolated proteins(e.g., isolated CoaX proteins encoded, for example, by any one of thecoaX genes or nucleic acids described herein). In one embodiment, theisolated proteins are produced by recombinant DNA techniques and can beisolated from microorganisms expressing, for example, any one of thecoaX genes or nucleic acids described herein, by an appropriatepurification scheme using standard protein purification techniques. Inanother embodiment, proteins are synthesized chemically using standardpeptide synthesis techniques.

An “isolated” or “purified” protein (e.g., an isolated or purified CoaXenzyme) is substantially free of cellular material or othercontaminating proteins from the microorganism from which the protein isderived, or substantially free from chemical precursors or otherchemicals when chemically synthesized. In one embodiment, an isolated orpurified protein has less than about 30% (by dry weight) ofcontaminating protein or chemicals, more preferably less than about 20%of contaminating protein or chemicals, still more preferably less thanabout 10% of contaminating protein or chemicals, and most preferablyless than about 5% contaminating protein or chemicals.

A “partially purified” protein (e.g., a partially purified CoaX enzyme)is a composition comprising a protein of interest where the compositionhas been subjected to at least one purification step, separation step,concentration step, or the like, such that the protein of interest ispresent at a greater concentration or level than prior to thepurification step, separation step, concentration step, or the like. Inone embodiment, a partially purified protein has between about 50-65%(by dry weight) of contaminating protein or chemicals, preferablybetween about 40%-50% of contaminating protein or chemicals, morepreferably between about 30-40% of contaminating protein or chemicals.

Included within the scope of the present invention are CoaX proteinsencoded by naturally-occurring bacterial or microbial genes, forexample, by coaX genes derived from a microorganism selected from thegroup consisting of Aquifex aeolicus, Bacillus anthracis, Bacillushalodurans, Bacillus stearothermophilus, Bacillus subtilis, Caulobactercrescentus, Chlorobium tepidum, Clostridium acetobutylicum,Dehalococcoides ethenogenes, Deinococcus radiodurans, Desulfovibriovulgaris, Geobacter sulfurreducens, Pseudomonas putida, Rhodobactercapsulatus, Thiobacillus ferrooxidans, Streptomyces coelicolor,Synechocystis sp., Thermotoga maritima, Bordetella pertussis, Borreliaburgdorferi, Campylobacter jejuni, Clostridium difficile, Helicobacterpylori, Neisseria meningitidis, Neisseria gonorrhoeae, Porphyromonasgingivalis, Pseudomonas aeruginosa, Treponema pallidum, Xylellafastidiosa and Mycobacterium tuberculosis. Further included within thescope of the present invention are CoaX proteins that are encodedbacterial or microbial genes which differ from naturally-occurringbacterial or microbial genes described herein, for example, genes whichhave nucleic acids that are mutated, inserted or deleted, but whichencode proteins substantially similar to the naturally-occurring geneproducts of the present invention. For example, it is well understoodthat one of skill in the art can mutate (e.g., substitute) nucleic acidswhich, due to the degeneracy of the genetic code, encode for anidentical amino acid as that encoded by the naturally-occurring gene.Moreover, it is well understood that one of skill in the art can mutate(e.g., substitute) nucleic acids which encode for conservative aminoacid substitutions. It is further well understood that one of skill inthe art can substitute, add or delete amino acids to a certain degreewithout substantially affecting the function of a gene product ascompared with a naturally-occurring gene product, each instance of whichis intended to be included within the scope of the present invention.

In one embodiment, an isolated protein of the present invention isencoded by a coaX gene derived from a microorganism selected from thegroup consisting of Aquifex aeolicus, Bacillus anthracis, Bacillushalodurans, Bacillus stearothermophilus, Bacillus subtilis, Caulobactercrescentus, Chlorobium tepidum, Clostridium acetobutylicum,Dehalococcoides ethenogenes, Deinococcus radiodurans, Desulfovibriovulgaris, Geobacter sulfurreducens, Pseudomonas putida, Rhodobactercapsulatus, Thiobacillus ferrooxidans, Streptomyces coelicolor,Synechocystis sp., Thermotoga maritima, Bordetella pertussis, Borreliaburgdorferi, Campylobacter jejuni, Clostridium difficile, Helicobacterpylori, Neisseria meningitidis, Neisseria gonorrhoeae, Porphyromonasgingivalis, Pseudomonas aeruginosa, Treponema pallidum, Xylellafastidiosa and Mycobacterium tuberculosis. In another embodiment, anisolated protein of the present invention is encoded by a coaX genederived from a microorganism selected from the group consisting ofBacillus anthracis, Bordetella pertussis, Borrelia burgdorferi,Campylobacter jejuni, Clostridium difficile, Helicobacter pylori,Neisseria meningitidis, Neisseria gonorrhoeae, Porphyromonas gingivalis,Pseudomonas aeruginosa, Legionella pneumophila, Treponema pallidum andXylella fastidiosa (e.g., is encoded by a coaX gene derived from apathogenic bacteria). In yet another embodiment, an isolated protein ofthe present invention is encoded by a coaX gene derived from amicroorganism selected from the group consisting of Bordetellapertussis, Borrelia burgdorferi, Campylobacter jejuni, Clostridiumdifficile, Helicobacter pylori, Neisseria meningitidis, Pseudomonasaeruginosa, Treponema pallidum and Xylella fastidiosa (e.g., is encodedby a coaX gene derived from a pathogenic bacteria which has coaX as it'ssole pantothenate kinase encoding enzyme). In a preferred embodiment, anisolated protein of the present invention (e.g., a CoaX) has an aminoacid sequence as set forth in any one of SEQ ID NOs:SEQ ID NO:12, SEQ IDNO:70, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:2, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:3, SEQ ID NO:57, SEQ ID NO:8, SEQ IDNO:59, SEQ ID NO:7, SEQ ID NO:61, SEQ ID NO:6, SEQ ID NO:63, SEQ IDNO:4, SEQ ID NO:13, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:11, SEQ IDNO:21, SEQ ID NO:55, SEQ ID NO:14 or SEQ ID NO:67, SEQ ID NO:43 or SEQID NO:22, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:20, SEQ ID NO:10, SEQ IDNO:65 and SEQ ID NO:5. In other embodiments, an isolated protein of thepresent invention (e.g., a CoaX) is a homologue of the at least one ofthe proteins set forth as SEQ ID NOs:SEQ ID NO:12, SEQ ID NO:70, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:2, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:3, SEQ ID NO:57, SEQ ID NO:8, SEQ ID NO:59, SEQ IDNO:7, SEQ ID NO:61, SEQ ID NO:6, SEQ ID NO:63, SEQ ID NO:4, SEQ IDNO:13, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:11, SEQ ID NO:21, SEQ IDNO:55, SEQ ID NO:14 or SEQ ID NO:67, SEQ ID NO:43 or SEQ ID NO:22, SEQID NO:39, SEQ ID NO:41, SEQ ID NO:20, SEQ ID NO:10, SEQ ID NO:65 and SEQID NO:5 (e.g., comprises an amino acid sequence at least about 30-40%identical, preferably about 40-50% identical, more preferably about50-60% identical, and even more preferably about 60-70%, 70-80%, 80-90%,90-95% or more identical to the amino acid sequence of SEQ ID NOs:SEQ IDNO:12, SEQ ID NO:70, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:2, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:3, SEQ ID NO:57, SEQ IDNO:8, SEQ ID NO:59, SEQ ID NO:7, SEQ ID NO:61, SEQ ID NO:6, SEQ IDNO:63, SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:9, SEQ ID NO:15, SEQ IDNO:11, SEQ ID NO:21, SEQ ID NO:55, SEQ ID NO:14 or SEQ ID NO:67, SEQ IDNO:43 or SEQ ID NO:22, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:20, SEQ IDNO:10, SEQ ID NO:65 and SEQ ID NO:5, and has an activity that issubstantially similar to that of the protein encoded by the amino acidsequence of SEQ ID NOs:SEQ ID NO:12, SEQ ID NO:70, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:2, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:3, SEQ ID NO:57, SEQ ID NO:8, SEQ ID NO:59, SEQ ID NO:7, SEQ IDNO:61, SEQ ID NO:6, SEQ ID NO:63, SEQ ID NO:4, SEQ ID NO:13, SEQ IDNO:9, SEQ ID NO:15, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:55, SEQ IDNO:14 or SEQ ID NO:67, SEQ ID NO:43 or SEQ ID NO:22, SEQ ID NO:39, SEQID NO:41, SEQ ID NO:20, SEQ ID NO:10, SEQ ID NO:65 and SEQ ID NO:5,respectively.

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=# of identical positions/total # ofpositions×100), preferably taking into account the number of gaps andsize of said gaps necessary to produce an optimal alignment.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. A preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as inKarlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Suchan algorithm is incorporated into the NBLAST and XBLAST programs(version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al. (1997) Nucleic Acids Research25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov. Another preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller (1988)Comput Appl Biosci. 4:11-17. Such an algorithm is incorporated into theALIGN program available, for example, at the GENESTREAM network server,IGH Montpellier, FRANCE (http://vega.igh.cnrs.fr) or at the ISREC server(http://www.ch.embnet.org). When utilizing the ALIGN program forcomparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used.

In another preferred embodiment, the percent homology between two aminoacid sequences can be determined using the GAP program in the GCGsoftware package (available at http://www.gcg.com), using either aBlossom 62 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6,or 4 and a length weight of 2, 3, or 4. In yet another preferredembodiment, the percent homology between two nucleic acid sequences canbe accomplished using the GAP program in the GCG software package(available at http://www.gcg.com), using a gap weight of 50 and a lengthweight of 3.

VI. Recombinant Nucleic Acid Molecules, Vectors and Microorganisms

The present invention further features recombinant nucleic acidmolecules (e.g., recombinant DNA molecules) that include nucleic acidmolecules and/or genes described herein (e.g., isolated nucleic acidmolecules and/or genes), preferably pantothenate kinase-encoding genes(e.g., coaX genes). The present invention further features vectors(e.g., recombinant vectors) that include nucleic acid molecules (e.g.,isolated or recombinant nucleic acid molecules and/or genes) describedherein. In particular, recombinant vectors are featured that includenucleic acid sequences that encode bacterial gene products as describedherein, preferably bacterial nucleic acid sequences that encodebacterial pantothenate kinase proteins.

The term “recombinant nucleic acid molecule” includes a nucleic acidmolecule (e.g., a DNA molecule) that has been altered, modified orengineered such that it differs in nucleotide sequence from the nativeor natural nucleic acid molecule from which the recombinant nucleic acidmolecule was derived (e.g., by addition, deletion or substitution of oneor more nucleotides). Preferably, a recombinant nucleic acid molecule(e.g., a recombinant DNA molecule) includes an isolated nucleic acidmolecule or gene of the present invention (e.g., an isolated coaX gene)operably linked to regulatory sequences.

The term “recombinant vector” includes a vector (e.g., plasmid, phage,phasmid, virus, cosmid or other purified nucleic acid vector) that hasbeen altered, modified or engineered such that it contains greater,fewer or different nucleic acid sequences than those included in thenative or natural nucleic acid molecule from which the recombinantvector was derived. Preferably, the recombinant vector includes a coaXgene or recombinant nucleic acid molecule including such coaX gene,operably linked to regulatory sequences, for example, promotersequences, terminator sequences and/or artificial ribosome binding sites(RBSs), as defined herein.

The phrase “operably linked to regulatory sequence(s)” means that thenucleotide sequence of the nucleic acid molecule or gene of interest islinked to the regulatory sequence(s) in a manner which allows forexpression (e.g., enhanced, increased, constitutive, basal, attenuated,decreased or repressed expression) of the nucleotide sequence,preferably expression of a gene product encoded by the nucleotidesequence (e.g., when the recombinant nucleic acid molecule is includedin a recombinant vector, as defined herein, and is introduced into amicroorganism).

The term “regulatory sequence” includes nucleic acid sequences whichaffect (e.g., modulate or regulate) expression of other nucleic acidsequences. In one embodiment, a regulatory sequence is included in arecombinant nucleic acid molecule or recombinant vector in a similar oridentical position and/or orientation relative to a particular gene ofinterest as is observed for the regulatory sequence and gene of interestas it appears in nature, e.g., in a native position and/or orientation.For example, a gene of interest can be included in a recombinant nucleicacid molecule or recombinant vector operably linked to a regulatorysequence which accompanies or is adjacent to the gene of interest in thenatural organism (e.g., operably linked to “native” regulatorysequences, for example, to the “native” promoter). Alternatively, a geneof interest can be included in a recombinant nucleic acid molecule orrecombinant vector operably linked to a regulatory sequence whichaccompanies or is adjacent to another (e.g., a different) gene in thenatural organism. Alternatively, a gene of interest can be included in arecombinant nucleic acid molecule or recombinant vector operably linkedto a regulatory sequence from another organism. For example, regulatorysequences from other microbes (e.g., other bacterial regulatorysequences, bacteriophage regulatory sequences and the like) can beoperably linked to a particular gene of interest.

In one embodiment, a regulatory sequence is a non-native ornon-naturally-occurring sequence (e.g., a sequence which has beenmodified, mutated, substituted, derivatized, deleted including sequenceswhich are chemically synthesized). Preferred regulatory sequencesinclude promoters, enhancers, termination signals, anti-terminationsignals and other expression control elements (e.g., sequences to whichrepressors or inducers bind and/or binding sites for transcriptionaland/or translational regulatory proteins, for example, in thetranscribed mRNA). Such regulatory sequences are described, for example,in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Regulatorysequences include those which direct constitutive expression of anucleotide sequence in a microorganism (e.g., constitutive promoters andstrong constitutive promoters), those which direct inducible expressionof a nucleotide sequence in a microorganism (e.g., inducible promoters,for example, xylose inducible promoters) and those which attenuate orrepress expression of a nucleotide sequence in a microorganism (e.g.,attenuation signals or repressor sequences). It is also within the scopeof the present invention to regulate expression of a gene of interest byremoving or deleting regulatory sequences. For example, sequencesinvolved in the negative regulation of transcription can be removed suchthat expression of a gene of interest is enhanced.

In one embodiment, a recombinant nucleic acid molecule or recombinantvector of the present invention includes a nucleic acid sequence or genethat encodes at least one bacterial gene product (e.g., a gene productencoded by coaX) operably linked to a promoter or promoter sequence.Preferred promoters of the present invention include E. coli promotersor Bacillus promoters and/or bacteriophage promoters (e.g.,bacteriophage which infect E. coli or Bacillus). In one embodiment, apromoter is a Bacillus promoter, preferably a strong Bacillus promoter(e.g., a promoter associated with a biochemical housekeeping gene inBacillus or a promoter associated with a glycolytic pathway gene inBacillus). In another embodiment, a promoter is a bacteriophagepromoter. In a preferred embodiment, the promoter is from thebacteriophage SP01. In a particularly preferred embodiment, a promoteris the P₂₆ promoter set forth as SEQ ID NO:18 or the P₁₅ promoter setforth as SEQ ID NO:19. Additional preferred promoters include tef(thetranslational elongation factor (TEF) promoter) and pyc (the pyruvatecarboxylase (PYC) promoter), which promote high level expression inBacillus (e.g., Bacillus subtilis). Additional preferred promoters, forexample, for use in Gram positive microorganisms include, but are notlimited to, the amyE promoter or phage SP02 promoters. Additionalpreferred promoters, for example, for use in Gram negativemicroorganisms include, but are not limited to tac, trp, tet, trp-tet,lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, λ-P_(R) orλ-P_(L).

In another embodiment, a recombinant nucleic acid molecule orrecombinant vector of the present invention includes a terminatorsequence or terminator sequences (e.g., transcription terminatorsequences). The term “terminator sequences” includes regulatorysequences which serve to terminate transcription of a gene. Terminatorsequences (or tandem transcription terminators) can further serve tostabilize mRNA (e.g., by adding structure to mRNA), for example, againstnucleases.

In yet another embodiment, a recombinant nucleic acid molecule orrecombinant vector of the present invention includes sequences whichallow for detection of the vector containing said sequences (i.e.,detectable and/or selectable markers), for example, sequences thatovercome auxotrophic mutations, for example, trpC or leuB, etc.,fluorescent markers, and/or colorimetric markers (e.g.,lacZ/β-galactosidase), and/or antibiotic resistance genes (e.g., amp ortet).

In yet another embodiment, a recombinant nucleic acid molecule orrecombinant vector of the present invention includes an artificialribosome binding site (RBS). The term “artificial ribosome binding site(RBS)” includes a site within an MRNA molecule (e.g., coded within DNA)to which a ribosome binds (e.g., to initiate translation) which differsfrom a native RBS (e.g., a RBS found in a naturally-occurring gene) byat least one nucleotide. Preferred artificial RBSs include about 5-6,7-8, 9-10, 11-12, 13-14, 15-16, 17-18, 19-20, 21-22, 23-24, 25-26,27-28, 29-30 or more nucleotides of which about 1-2, 3-4, 5-6, 7-8,9-10, 11-12, 13-15 or more differ from the native RBS (e.g., the nativeRBS of a gene of interest). Preferably, nucleotides which differ aresubstituted such that they are identical to one or more nucleotides ofan ideal RBS for a particular gene. Artificial RBSs can be used toreplace the naturally-occurring or native RBS associated with aparticular gene. Artificial RBSs preferably increase translation of aparticular gene.

In another embodiment, a recombinant vector of the present inventionincludes sequences that enhance replication in bacteria (e.g.,replication-enhancing sequences). In one embodiment,replication-enhancing sequences are derived from E. coli. In anotherembodiment, replication-enhancing sequences are derived from pBR322.

In yet another embodiment, a recombinant vector of the present inventionincludes antibiotic resistance genes. The term “antibiotic resistancegenes” includes sequences which promote or confer resistance toantibiotics on the host organism. In one embodiment, the antibioticresistance genes are selected from the group consisting of cat(chloramphenicol resistance) genes, tet (tetracycline resistance) genes,amp (ampicillin resistence), erm (erythromycin resistance) genes, neo(neomycin resistance) genes and spec (spectinomycin resistance) genes.Recombinant vectors of the present invention can further includehomologous recombination sequences (e.g., sequences designed to allowrecombination of the gene of interest into the chromosome of the hostorganism). For example, amyE sequences can be used as homology targetsfor recombination into the host chromosome.

Preferred vectors of the present invention include, but are not limitedto, vectors set forth in FIGS. 8-10. It will further be appreciated byone of skill in the art that the design of a vector can be tailoreddepending on such factors as the choice of microorganism to begenetically engineered, the level of expression of gene product desiredand the like.

The methodologies of the present invention feature microorganisms, e.g.,recombinant microorganisms, preferably including genes or vectors asdescribed herein, in particular, pantothenate kinase encoding genes orvectos. The term “recombinant” microorganism includes a microorganism(e.g., bacteria, yeast cell, fungal cell, etc.) which has beengenetically altered, modified or engineered (e.g., geneticallyengineered) such that it exhibits an altered, modified or differentgenotype and/or phenotype (e.g., when the genetic modification affectscoding nucleic acid sequences of the microorganism) as compared to thenaturally-occurring microorganism from which it was derived. Preferably,a “recombinant” microorganism of the present invention has beengenetically engineered such that it overexpresses at least one bacterialgene or gene product (e.g., a pantothenate kinase encoding gene) asdescribed herein, preferably a pantothenate kinase encoding-geneincluded within a recombinant vector as described herein. The ordinaryskilled will appreciate that a microorganism expressing oroverexpressing a gene product produces or overproduces the gene productas a result of expression or overexpression of nucleic acid sequencesand/or genes encoding the gene product.

The term “overexpressed” or “overexpression” includes expression of agene product (e.g., a pantothenate kinase) at a level greater than thatexpressed prior to manipulation of a microorganism or in a comparablemicroorganism that has not been manipulated. In one embodiment, amicroorganism is genetically manipulated (e.g., genetically engineered)to overexpress a level of gene product greater than that expressed priorto manipulation of the microorganism or in a comparable microorganismwhich has not been manipulated. Genetic manipulation can include, but isnot limited to, altering or modifying regulatory sequences or sitesassociated with expression of a particular gene (e.g., by adding strongpromoters, inducible promoters or multiple promoters or by removingregulatory sequences such that expression is constitutive), modifyingthe chromosomal location of a particular gene, altering nucleic acidsequences adjacent to a particular gene such as a ribosome binding siteor transcription terminator, increasing the copy number of a particulargene, modifying proteins (e.g., regulatory proteins, suppressors,enhancers, transcriptional activators and the like) involved intranscription of a particular gene and/or translation of a particulargene product, or any other conventional means of deregulating expressionof a particular gene routine in the art (including but not limited touse of antisense nucleic acid molecules, for example, to blockexpression of repressor proteins).

In another embodiment, the microorganism can be physically orenvironmentally manipulated to overexpress a level of gene productgreater than that expressed prior to manipulation of the microorganismor in a comparable microorganism which has not been manipulated. Forexample, a microorganism can be treated with or cultured in the presenceof an agent known or suspected to increase transcription of a particulargene and/or translation of a particular gene product such thattranscription and/or translation are enhanced or increased.Alternatively, a microorganism can be cultured at a temperature selectedto increase transcription of a particular gene and/or translation of aparticular gene product such that transcription and/or translation areenhanced or increased.

Still other preferred recombinant microorganisms of the presentinvention are mutant microorganisms. As used herein, the term “mutantmicroorganism” includes a recombinant microorganism that has beengenetically engineered to express a mutated gene or protein that isnormally or naturally expressed by the microorganism. Preferably, amutant microorganism expresses a mutated gene or protein such that themicroorganism exhibits an altered, modified or different phenotype(e.g., has been engineered to express a mutated CoaA biosyntheticenzyme, for example, pantothenate kinase). In one embodiment, a mutantmicroorganism is designed or engineered such that it includes a mutantcoaX gene, as defined herein. In another embodiment, a recombinantmicroorganism is designed or engineered such that it includes a mutantcoaA gene, as defined herein. In another embodiment, a mutantmicroorganism is designed or engineered such that a coaX gene has beendeleted (i.e., the protein encoded by the coaX gene is not produced). Inanother embodiment, a mutant microorganism is designed or engineeredsuch that a coaA gene has been deleted (i.e., the protein encoded by thecoaA gene is not produced). Preferably, a mutant microorganism has amutant coaX gene or a mutant coaA gene, or has been engineered to have acoaX gene and/or coaA deleted, such that that the mutant microorganismencodes a “reduced pantothenate kinase activity”. In the context of awhole microorganism, pantothenate kinase activity can be determined bymeasuring or assaying for a decrease in an intermediate or product ofthe CoA biosynthetic pathway, for example, measuring or assaying for4′-phosphopantothenate, 4′-phosphopantothenylcysteine,4′-phosphopantetheine, dephosphocoenzyme A, Coenzyme A, apo-acyl carrierprotein (apo-ACP) or holo-acyl carrier protein (ACP) in themicroorganism (e.g., in a lysate isolated or derived from themicroorganism) or in the medium in which the microorganism is cultured.Alternatively, pantothenate kinase or CoaX activity can be determined bymeasuring or assaying for increased or decreased growth of themicroorganism. Alternatively, pantothenate kinase activity can bedetermined indirectly by measuring or assaying for an increase inpantothenate which is the immediate precursor of pantothenate kinase.

In one embodiment, a recombinant microorganism of the present inventionis a Gram negative organism (e.g., a microorganism which excludes basicdye, for example, crystal violet, due to the presence of a Gram-negativewall surrounding the microorganism). In another embodiment, arecombinant microorganism of the present invention is a Gram positiveorganism (e.g., a microorganism which retains basic dye, for example,crystal violet, due to the presence of a Gram-positive wall surroundingthe microorganism). In a preferred embodiment, the recombinantmicroorganism is a microorganism belonging to a genus selected from thegroup consisting of Escherichia, Heliobacter, Pseudomonas, Bordetellaand Bacillus. In a more preferred embodiment, the recombinantmicroorganism is of the genus Escherichia or Bacillus.

In another embodiment, the recombinant microorganism is a Gram negative(excludes basic dye) organism. In a preferred embodiment, therecombinant microorganism is a microorganism belonging to a genusselected from the group consisting of Salmonella, Escherichia,Klebsiella, Serratia, and Proteus. In a more preferred embodiment, therecombinant microorganism is of the genus Escherichia. In an even morepreferred embodiment, the recombinant microorganism is Escherichia coli.In another embodiment, the recombinant microorganism is Saccharomyces(e.g., S. cerevisiae).

V. Screening Assays

Because CoaX is an essential factor in bacteria, proteins (e.g.,enzymes) involved in the biosynthesis of CoA provide valuable tools inthe search for novel antibiotics. In particular, the CoaX protein is avaluable target for identifying bacteriocidal compounds because it bearsno resemblance in primary sequence to mammalian pantothenate kinaseenzymes or CoaA's that are essential for beneficial enteric bacteriasuch as E. coli. Accordingly, the present invention also provides amethod (also referred to herein as a “screening assay”) for identifyingmodulators, i.e., candidate or test compounds or agents (e.g., peptides,peptidomimetics, small molecules or other drugs) that bind to CoaX, orhave a stimulatory or inhibitory effect on, for example, coaX expressionor CoaX activity.

In one embodiment, the invention provides assays for screening candidateor test compounds that are capable of binding to CoaX proteins or abiologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compounds thatmodulate the activity of CoaX proteins or biologically active portionsthereof. As used herein, the phrase “CoaX” activity includes anydetectable or measurable activity of the CoaX protein, i.e., the proteinencoded by the coaX gene of the present invention, for example, the coaXgene derived from a microorganism selected from the group consisting ofAquifex aeolicus, Bacillus anthracis, Bacillus halodurans, Bacillusstearothermophilus, Bacillus subtilis, Caulobacter crescentus,Chlorobium tepidum, Clostridium acetobutylicum, Dehalococcoidesethenogenes, Deinococcus radiodurans, Desulfovibrio vulgaris, Geobactersulfurreducens, Pseudomonas putida, Rhodobacter capsulatus, Thiobacillusferrooxidans, Streptomyces coelicolor, Synechocystis sp., Thermotogamaritima, Bordetella pertussis, Borrelia burgdorferi, Campylobacterjejuni, Clostridium difficile, Helicobacter pylori, Neisseriameningitidis, Neisseria gonorrhoeae, Porphyromonas gingivalis,Pseudomonas aeruginosa, Treponema pallidum, Xylella fastidiosa,Legionella pneumophila, and Mycobacterium tuberculosis. In a preferredembodiment, a CoaX activity is at least one of the following: (1)modulation of at least one step in the CoA biosynthetic pathway; (2)promotion of CoA biosynthesis; ( 3 ) phosphorylation of a CoaXsubstrate; ( 4 ) a pantothenate kinase activity; and ( 4 )complementation of a CoaX mutant.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in chemical compound library methods known inthe art, including: natural compound libraries; biological libraries;spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the ‘one-beadone-compound’ library method; and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to peptide libraries, while the other approaches are applicableto peptide, non-peptide oligomer or small molecule libraries ofcompounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. AcadSci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med Chem. 37:1233. Libraries of compoundsmay be presented in solution (e.g., Houghten (1992) Biotechniques13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor(1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409),spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al. (1992) ProcNatl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990)Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla etal. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol.Biol. 222:301-310); (Ladner supra.).

In one embodiment, an assay is a microorganism-based assay in which arecombinant microorganism that expresses a CoaX protein or biologicallyactive portion thereof is contacted with a test compound and the abilityof the test compound to modulate CoaX activity is determined.Determining the ability of the test compound to modulate CoaX activitycan be accomplished by monitoring, for example, growth, intracellularphosphopanthoate or CoA concentrations, or secreted pantothenateconcentrations (as compounds that inhibit CoaX will result in a buildupof pantothenate in the test microorganism). CoaX substrate can belabeled with a radioisotope or enzymatic label such that modulation ofCoaX activity can be determined by detecting a conversion of labeledsubstrate to intermediate or product. For example, CoaX substrates canbe labeled with ³²P, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radio emmission or byscintillation counting. Determining the ability of a compound tomodulate CoaX activity can alternatively be determined by detecting theinduction of a reporter gene (comprising a CoA-responsive regulatoryelement operatively linked to a nucleic acid encoding a detectablemarker, e.g., luciferase), or detecting a CoA-regulated cellularresponse.

In yet another embodiment, a screening assay of the present invention isa cell-free assay in which a CoaX protein or biologically active portionthereof is contacted with a test compound in vitro and the ability ofthe test compound to bind to or modulate the activity of the CoaXprotein or biologically active portion thereof is determined. In apreferred embodiment, the assay includes contacting the CoaX protein orbiologically active portion thereof with known substrates to form anassay mixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to modulate enzymaticactivity of the CoaX on its substrates.

Screening assays can be accomplished in any vessel suitable forcontaining the microorganisms, proteins, and/or reactants. Examples ofsuch vessels include microtiter plates, test tubes, and micro-centrifugetubes. In more than one embodiment of the above assay methods of thepresent invention, it may be desirable to immobilize either CoaXprotein, CoaX substrate, substrate analogs or a recombinantmicroorganism expressing CoaX protein to facilitate separation ofproducts, ligands, and/or substrates, as well as to accommodateautomation of the assay. For example, glutathione-S-transferase/CoaXfusion proteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtiter plates.Other techniques for immobilizing proteins on matrices (e.g.,biotin-conjugation and streptavidin immobilization or antibodyconjugation) can also be used in the screening assays of the invention.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, a CoaX modulating agentidentified as described herein (e.g., an anti-bactericidal compound) canbe used in an infectious animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.

CoaX modulators can further be designed based on the crystal structureof any one of the CoaX proteins of the present invention. In particular,based at least in part on the discovery of CoaX as an essentialbacterial protein, one can produce significant quantities of the CoaXprotein, for example using the recombinant methodologies as describedherein, purify and crystallize said protein, subject said protein toXray crystallographic procedures and, based on the determined crystalstructure, design modulators (e.g., active site modulators, for example,competitor molecules, active site inhibitors, and the like), and testsaid designed modulators according to any one of the assays describedherein.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES Example I Assays for CoaX Genes or Activities Assay forPantothenate Kinase Genes or In Vivo Pantothenate Kinase Activity

In order to assay for genes encoding pantothenate kinase, the ability ofplasmids containing these genes to complement the coaA15(Ts15) mutationin E. coli strain YH1 is tested at the non-permissive temperature of43°-44° C. The defect in E. coli coaA15(Ts) has been identified as anS177L mutation that lies in a region that is highly conserved amongbacterial pantothenate kinases, including CoaA of B. subtilis. StrainYH1 was constructed by P1 transduction from publically available strainDV62 (Coli Genetic Stock Center) to publically available strain YMC9(ATCC), selecting for tetracycline resistance and screening fortemperature sensitivity at 43° C.

In Vitro Assay for Pantothenate Kinase Activity

The assay for pantothenate kinase is based on the fact that underappropriate mildly acidic conditions (1% acetic acid in 95% ethanol),the product of the reaction, 4′-phosphopantothenate, binds to positivelycharged ion exchange paper, while the substrate, pantothenate, does not(see Vallari, D., Jackowski, S., and Rock, C., (1987), Journal ofBiological Chemistry, Vol. 262, pp2468-2471, hereby incorporated byreference).

Cells of the strain to be assayed (bacteria, yeast, fungi, animal, orplant cells) are grown to late logarithmic phase or stationary phase, in200 ml of an appropriate medium, for example Luria Broth or M9 minimalsalts plus 0.5% glucose plus any necessary additives (for bacterialcells), at an appropriate temperature (25 to 44° C.). All subsequentsteps are carried out at 0 to 40° C. The culture is cooled on ice for 10minutes and the cells are concentrated by centrifugation at 7,000×g for10 minutes. The cell pellet is rinsed by resuspending it in ice coldBuffer A (50 mM Tris-HCl, pH 7.4, 2.5 mM MgCl₂) and recentrifugation.

The rinsed cells are resuspended in the minimum possible volume (2-5 ml,depending on the size of the pellet) of Buffer A. The cells are thenbroken open by sonication in an inverted stainless steel test tube capon ice for four bursts of 15 seconds each with 30 seconds of coolingbetween each burst. Cell debris is then removed from the lysed cells bycentrifugation at 10,000×g for 10 minutes. The supernatant solution isthen dialyzed for 12-16 hrs against two changes of one liter of Buffer Awith 0.1 mM dithiothreitol added. Dialysis may be necessary to preventthe reaction product from undergoing further reactions catalyzed by thecrude cell extract. Protein concentration in the dialyzed extracts ismeasured with a BCA Protein Assay Kit from BioRad.

The assay mix contains (final amounts or concentrations) about zero to150 μg protein, 80 μM ¹⁴C-D-pantothenate, specific activity about 60,000dpm/nmole (purchased from American Radiolabeled Chemicals, Inc.), 2.5 mMATP (Sigma Chemical Company, sodium salt), 2.5 mM MgCl2, and 100 mM TrisHCl, pH 7.4, in a total volume of 40 μl. The reaction mix, minus theATP, can be preincubated for about 1 to 10 minutes at an appropriatetemperature (25 to 55° C.), in which case the reaction is started byaddition of the ATP from a concentrated stock, also preincubated at theassay temperature.

After incubation for 1 to 10 minutes, the reaction is stopped bypipetting 35 μl of the reaction mix into an Eppendorf tube containing 1ml of 95% ethanol, 1% acetic acid. After thorough mixing, theprecipitated protein is pelleted in a microcentrifuge at top speed forone minute. The resulting supernatant solution is then applied to a oneinch (or other appropriate size) disk of Whatman DE81 ion exchangefilter paper prewetted with distilled water in a vacuum filtrationmanifold (for example Millipore 1225 Sampling Manifold). Each disk isthen rinsed three times with 10 ml of 1% acetic acid in 95% ethanol. Thetop plate is then removed from the manifold and the completely exposedfilter disks are each rinsed once more with 5 ml of the same rinsesolution. The rinsed filters are then counted in a scintillation counterappropriatly set for ¹⁴C in 10 ml of Ecolume scintillation fluid. Thespecific activity of the pantothenate kinase can be calculated bydetermining the number of moles of substrate converted to product per mgprotein per minute under various appropriate conditions of the assay.

Appropriate assay blanks include, but are not limited to, the standardmix except without ATP or without protein extract, or a complete mixincubated on ice for the shortest possible time before pipetting to thefilter disk (preferably less than 10 seconds).

The assay should be checked for linearity with time up to 10 minutes,and for linearity with protein between zero and 150 μg. No more than 10%of the input 14C-pantothenate should be converted to phosphorylatedproduct for the most accurate measurement of activity.

Temperature sensitivity of the pantothenate kinase enzyme can be testedby preincubating the reaction mix at various temperatures (25 to 55° C.)for various lengths of time (zero to 60 minutes) before addition of ATPto start the reaction.

For pantothenate kinases other than that encoded by the E. coli coaAgene, the optimum temperature, pH, MgCl₂ concentration, buffering ion,ATP (or other substrate containing a high energy phosphate donor)concentration, salt type and concentration, total ionic strength, etc.,may need to be determined. For accurate determination of enzymeactivity, it may be necessary to purify or partially purify thepantothenate kinase enzyme from crude extracts, for example by ammoniumsulfate fractionation and/or by column chromatography.

The assay may be adapted for high throughput screening, for example byusing γ-thio-ATP instead of ATP and then reacting the transfered thiogroup with a conveniently detectable signalling molecule (see Jeong, S.,and Nikiforov, T., (1999), Biotechniques Vol. 27, pp 1232-1238; andFacemyer, K., and Cremo, C., (1992), Bioconjug. Chem. Vol. 3, pp408-413, both of which are hereby incorporated by reference).

Example II Identification and Characterization of a First B. subtilisGene Encoding Pantothenate Kinase, the coaA Gene

The annotated version of the B. subtilis genome sequence available onthe “Subtilist” web site contained no gene labeled as coaA. However ahomology search using the protein sequence of E. coli pantothenatekinase as a query sequence gave a good match with B. subtilis gene yqjS,which is annotated as “unknown; similar to pantothenate kinase.” Thisgene appears to be the penultimate gene in an operon containing fiveopen reading frames (FIG. 2). Two of the open reading frames encodeproteins which are similar to D-serine dehydratase and to “ketoacylreductase”; the other two have no known homologies. For the open readingframe corresponding to coaA, there are three possible start codons; eachhaving a possible ribosome-binding site (RBS) associated with it. Thethree potential coaA ORFs were named coaA1, coaA2, and coaA3, fromlongest to shortest.

All three potential coaA open reading frames were cloned along withtheir respective RBSs by PCR followed by ligation into expressionplasmid pAN229 to form plasmids pAN281, pAN282 and pAN283. pAN229 is alow copy vector in E. coli that provides expression from the SP01 phageP₁₅ promoter and can integrate by single crossover at bpr withtetracycline selection.

To determine if the cloned putative coaA ORFs actually encode apantothenate kinase activity, several isolates of all three plasmidswere transformed into the E. coli strain YH1, that contains thecoaA15(Ts) allele. Transformants were streaked to plates incubated at30° and 43° C. to test for complementation of the temperature sensitiveallele. Isolates of all three coaA variants complemented well at 43° C.,indicating that all three plasmid constructs encode an activepantothenate kinase. Accordingly, it can be concluded that the B.subtilis yqjS open reading frame codes for an active pantothenatekinase.

Example III Deletion of the coaA Gene From the B. subtilis genome

The coaA gene of B. subtilis (yqjs) was deleted from the chromosome of aB. subtilis strain by conventional means. The majority of the coaAcoding sequence was deleted from a plasmid clone and replaced by achloramphenicol resistance gene (cat), while leaving approximately 1 kbof upstream and downstream sequence to allow homologous recombinationwith the chromosome, to give plasmid pAN296 (see FIG. 3). pAN296 wasthen used to transform a B. subtilis strain (PY79), selecting forchloramphenicol resistance. The majority of transformants result from adouble crossover event that effectively substitutes the cat gene for thecoaA gene. The transformed strain containing the coaA deletion—catinsertion, named PA861) grew normally indicating the presence of asecond B. subtilis pantothenate kinase encoding gene described herein.

Example IV Identification and Characterization of a Second B. subtilisGene Encoding Pantothenate Kinase Activity, the coaX Gene

After finding that deletion of the coaA gene from the chromosome of B.subtilis is not a lethal event (see Example III), it was concluded thatB. subtilis must contain a second gene that encodes an activepantothenate kinase, since pantothenate kinase is an essential enzymeactivity.

A second pantothenate kinase-encoding gene was identified bycomplementing the E. coli strain YH1 (coaA15(Ts)) with a B. subtilisgene bank and selecting for transformants that were able to grow at 43°C. Found among the transformants were two families of plasmids that hadoverlapping restriction maps within each family, but not between thefamilies. As expected, the restriction map of one family was identicalto that predicted from the B. subtilis genome sequence for the homologueof the E. coli coaA gene (which we named coaA also, see above) andsurrounding sequences. The other family had a restriction map that wascompletely non-overlapping with the first.

DNA sequencing of the ends of the cloned inserts from the second familyshowed that the clones came from a region of the B. subtilis chromosomethat includes the 3′ end of the ftsH gene, the 5′ end of the sul gene,and all of the yacB, yacC, yacD, cysK, pabB, pabA and pabC genes. Noneof the open reading frames of these cloned inserts showed homology toany known pantothenate kinase sequences, either prokaryotic oreukaryotic.

Several deletions were created through the B. subtilis genomic sequencesin the cloned inserts. Each deletion was tested for complementation ofthe E. coli temperature sensitive pantothenate kinase. In particular, adeletion that removed all DNA between a Stu I site in the cloning vectorand a Swa I site in the yacC gene, leaves yacB as the only intact openreading frame in the cloned insert (see FIG. 4). This deleted plasmidstill complemented the E. coli pantothenate kinase mutant. However,another deletion that removed DNA from the Swa I site in yacC through aBst11071 site in the (already truncated) ftsH gene, could not complementthe E. coli pantothenate kinase mutant. From these results, it wasconcluded that the yacB open reading frame was responsible for thecomplementation activity. To confirm that yacB is a pantothenate kinasegene, the yacB ORF plus 112 base pairs of downstream flanking sequencewas amplified by PCR in two independent reactions and cloned downstreamof a constitutive promote to give plasmids pAN341 and pAN342 (FIG. 5).Both pAN341 and pAN342 complemented the defect in YH1 at 44° C., while acontrol plasmid, which has the same backbone, but expresses panBCDinstead of yacB did not. This confirmed that the yacB open reading framewas responsible for the complementation of YH1.

As such, a novel gene that encodes pantothenate kinase activity in B.subtilis has been discovered that is not related by homology to anypreviously known pantothenate kinase gene. This gene has been renamedcoaX, as a second, alternative gene that encodes an enzyme thatcatalyzes the first step in the pathway from pantothenate to CoaA. In B.subtilis strains deleted for coaA, coaX is an essential gene.

Several homologues of the B. subtilis coaX gene were identified byhomology searching of various publically available databases using thepublished yacB (coaX) open reading frame sequence and predicted aminoacid sequence (as set forth in SEQ ID NOs:15 and 16 respectively). Intwo cases (Mycobacterium tuberculosis and Streptomyces coelicolor) thehomologous coaX genes are adjacent to, or almost adjacent to,pantothenate biosynthetic genes, consistent with these homologs having arole in pantothenate metabolism. The CoaX proteins show no homology tothe CoaA family of pantothenate kinases, nor to the eukaryotic family ofpantothenate kinases exemplified by PanK of Saccharomyces cerevisiae.

Alignment of the amino acid sequences of several bacterial CoaX homologswith the amino acid sequence predicted from translating the B. subtilisyacB ORF described in the published B. subtilis genome sequence revealedthat the CoaX proteins from other bacteria contained additional aminoacid residues at their carboxy-terminal ends. Moreover, these extensionsbeyond the end of the predicted amino acid sequence for the B. subtilisgene product contained two relatively well conserved segments ofsequence.

Translation of nucleotide sequences just downstream from the stop codonof the B. subtilis yacB ORF in a different reading frame revealed theexistence of amino acid sequences very similar to the carboxy-terminalextensions of the other bacterial CoaX proteins. It is thus believedthat an error exists in the published DNA sequence of the B. subtilisyacB ORF sequence that causes a frame shift leading to an artifactualdownstream amino acid sequence and premature termination.

The PCR-generated sequences of B. subtilis coaX in pAN341 and pAN342(described above) contain enough downstream flanking sequence to encodethe putative carboxy-terminal extension described above, which isconsistent with the result that the clones were functional in thecomplementation assay. However when the 3′ PCR primer was positioned toinclude only the shorter yacB ORF predicted from the published sequence,but not to include the putative carboxy-terminal extension, then theresulting plasmids, pAN329 and pAN330 (similar in structure to pAN341and pAN342; see FIG. 5), did not complement the defect in YH1. Thisresult supports the notion that the published yacB coding sequencecontains a frame-shift error, and that the carboxy-terminal end of CoaXis necessary for pantothenate kinase activity. A predicted correctnucleotide sequence for B. subtilis coaX is set forth as SEQ ID NO:1 andthe translated amino acid sequence is set forth as SEQ ID NO:2. Amultiple sequence alignment of the CoaX amino acid sequences of B.subtilis and 11 homologues thereof is set forth in FIG. 6.

Example V Deleting the Second Pantothenate Kinase Gene, coaX Gene FromB. subtilis

With the knowledge gained above concerning the existence and nature ofcoaX, one can create a deletion of the coaX open reading frame from theB. subtilis chromosome that will remove the encoded activity, and thatwill not adversely affect the expression of the genes downstream fromcoaX. In such a deleted strain, the coaA gene will be the only gene thatencodes pantothenate kinase.

To delete the coaX gene from B. subtilis, plasmid pAN336, which containsupstream and downstream homology for double crossover, was constructedwith a kanamycin resistance gene replacing most of the coaX ORF (FIG.7). Strain PY79 was transformed to kanamycin resistance by pAN336, andan isolate confirmed to have resulted from a double crossover by PCR wasnamed PA876. As predicted, deletion of coaX by itself is not lethal forB. subtilis. Furthermore, chromosomal DNA from PA876 would not transformcompetent PA861 (PY79 ΔcoaA::cat) to kanamycin resistance. These resultsindicate that it is the combination of ΔcoaA::cat and ΔcoaX::kan that islethal for B. subtilis, confirming that B. subtilis contains twounlinked genes that encode pantothenate kinase, coaA and coaX, and thateither gene alone is capable of supplying sufficient pantothenate kinasefor a normal rate of growth.

Example VI Identification of coaX Homologs in Other Microbes

Database analyses reveal that many bacteria, in addition to B. subtilis,contain homologs of the CoaX pantothenate kinase. As shown in Tables 1and 2, both nonpathogenic and pathogenic bacteria can be found thatcontain homologs of this novel gene.

TABLE 1 CoaX homologs in Non-Pathogens Genome CoaA Species completehomolog CoaX homolog Aquifex aeolicus Yes NONE RAA00700 aq_1924AAC07720.1 pir||E70465 Bacillus halodurans Yes BH2875 BH0086 BAB06594.1Bacillus No NONE? gnl|UOKNOR_1422|bstear_.Contig467 stearothermophilusBacillus subtilis Yes RBS02372 YqjS RBS00070 YacB BAA12625.1 BAA05305.1CAB14308.1 CAB11846.1 pir||C69965 pir||S66100 Caulobacter crescentus NoNONE? gnl|TIGR|C.crescentus_12574 Chlorobium tepidum No NONE?gnl|TIGR|C.tepidum_3499 Clostridium No NONE? RCA03301 acetobutylicumgnl|GTC|C.aceto_gnl Dehalococcoides No NONE? gnl|TIGR_61435|deth_1587ethenogenes Deinococcus Yes NONE AAF10040.1 radiodurans pir||E75516Desulfovibrio vulgaris No NONE? BAA21476.1 P37564gnl|TIGR_881|dvulg_1371 Geobacter No NONE? gnl|TIGR_35554|gsulf_121sulfurreducens Pseudomonas putida No NONE? gnl|TIGR|pputida_10724 KT2440Rhodobacter No NONE? RRC02473 capsulatus Thiobacillus No NONE?gnl|TIGR|t_ferrooxidans_6155 ferrooxidans Streptomyces No COAA_STRCOSCE94.31c coelicolor g8469186 CAB40880.1 pir||T35567 Synechocystis sp.Yes NONE ORF_ID:slr0812 BAA18120 Thermotoga maritima Yes NONE TM0883AAD35964.1 pir||D72320

TABLE 2 CoaX homologs in Pathogens Genome CoaA Pathogen complete homologCoaX homolog Comments Haemophilus Yes RHI13313 NONE influenzaeStreptococcus No RST01295 NONE pyogenes Yersinia pestis No RYP02180 NONEVibrio cholerae Yes VC0320 NONE Bacillus anthracis No NONE? YESBordetella No NONE? BAF (BVG ACCESSORY pertussis FACTOR) Borrelia YesNONE BB0527 burgdorferi Campylobacter Yes NONE Cj0394c jejuniClostridium No NONE? YES difficile Helicobacter Yes NONE jhp0796 (strainJ99) pylori HP0862 (strain 26695) AAD07916.1 Neisseria Yes NONE NMA0357(strain Z2491) CoaX is fused meningitidis NMB2075 (strain MC58) to BirANeisseria No NONE? RNG00193 CoaX is fused gonorrhoeae to BirAPorphyromonas No NONE? RPG01037 gingivalis gnl|TIGR|P.gingivalis_GPG.conPseudomonas Yes NONE RPA06755 aeruginosa PA4279 AAG07667.1 Treponema YesNONE RTP00155 (TP0431) pallidum Xylella fastidiosa Yes NONE XF1795Legionella No gnl|CUCGC_446|lpneumo_C030598.2F12.S pneumophilaMycobacterium No MLCB1222.23 leprae Mycobacterium Yes RMT04257 RMT02984(Rv3600c) RMT04257 tuberculosis

Of particular interest are the seven human pathogens Helicobacterpylori, Borrelia burgdorferi, Pseudomonas aeruginosa, Campylobacterjejuni, Neisseria meningitidis, Treponema pallidum, and Bordetellapertussis, that contain the CoaX pantothenate kinase as their solepantothenate kinase activity. For these bacteria, the CoaX pantothenatekinase represents an attractive target for screening for new antibioticseffective against one or more of these pathogens. One can overproducethe particular CoaX pantothenate kinase and use the isolated protein,partially purified protein or crude cell extracts to screen in vitro forcompounds that modulate (e.g. inhibit) the pantothenate kinase activity.Alternatively, one can isolate compounds that specifically bind to theenzyme and test their ability to block the enzyme's activity. A knownkinase activity represents a particularly favorable target forhigh-throughput screening for compounds that modulate or decrease thatactivity.

Also of interest are other pathogens which contain a coaX gene, inparticular, if it is demonstrated that these other pathogens containonly a single pantothenate kinase encoded by the coaX gene. Examples ofsuch bacteria are Porphyromonas gingivalis, Neisseria gonorrhoeae,Clostridium difficile, and Bacillus anthracis, all of which have beenshown to contain a coaX homolog. Determination whether or not they alsocontain a second pantothenate kinase encoded by a coaA homolog can bedetermined according to the methodologies taught in Examples II-IV.

Example VII Identification of coaX Homologs in Human Pathogens Lacking aConventional Prokaryotic Pantothenate Kinase

Human pathogens Helicobacter pylori (agent in gastoenteritus, stomachulcers, and potentially stomach cancer), Borrelia burgdorferi (agent inLyme's disease), Bordetella pertussis (agent in whooping cough), andPseudomonas aeruginosa (opportunistic pathogen in cystic fibrous) allcontain homologs of the coaX gene of B. subtilis and no homologs of thecoaA gene of E. coli or B. subtilis. This is also true for the pathogensTreponema pallidum, Campylobacter jejuni, and Neisseria meningitidis. Wehave shown in B. subtilis that in the absence of the coaA gene product(ΔcoaA mutant), the coaX gene product is essential, providing the onlypantothenate kinase activity required for the synthesis of the essentialcompound, Coenzyme A. Therefore it can be predicted that thepantothenate kinase encoded by the coaX homolog in the above listedpathogens is an essential enzyme for each mentioned pathogen and isrequired for the survival and growth of the pathogen. In fact it hasbeen reported that the coaX homolog in Bordetella pertussis, called baf,and classified as an auxiliary regulatory factor rather than a criticalenzyme, is an essential gene (see Wood, G. E. and R. L. Friedman (2000)FEMS Microbial. Lett. 193(1):25-30).

The CoaX protein is a favorable target for the development and screeningof new antibiotics. First, the pantothenate kinase encoded by the coaXgene is an essential enzyme in a group of human pathogens, making it agood target for inactivation. Second, the enzyme activity (kinase) ofthe isolated CoaX protein or its homologs provides an ideal assay toscreen large numbers of compounds (combinatorial libraries, etc.) fortheir ability to specifically inhibit the pantothenate kinase activityboth in vitro and in vivo.

Example VIII Production of CoaX Proteins From Pathogens for Use inScreening Assays.

To provide the pantothenate kinase proteins for screening assays, thecoaX gene homolog was obtained by PCR from isolated, whole genome DNA ofHelicobacter pylori (ATCC 700392), Borrelia burgdorferi (ATCC 35210),Bordetella pertussis (ATCC 9797), and Pseudomonas aeruginosa (ATCC47085). Coding sequences for proteins with homology to B. subtilis CoaXwere amplified by PCR using the primers and templates given in Table 3with Pfx DNA polymerase (Life Technologies) according to themanufacture's specifications. The PCR primers incorporate a XbaIrestriction enzyme recognition site at the 5′ end of each product and aBamHI restriction enzyme recognition site at the 3′ end of each product.PCR products were digested with a mixture of XbaI and BamHI and thenpurified by preparative agarose gel electrophoresis.

TABLE 3 PCR primers and template DNAs used to amplIfy coding sequenceshomologous to B. subtilis coaX. coaX Template 5′ amplification3′ amplification Organism homolog DNA primer primer Bacillus yacB StrainRL-1 TP175 TP176 subtilis 168 genomic DNA 5′-GGGTCTA 5′-GGGATCCGAAAAGGAGG TTATACACTT AATTTAAATG CCTACGCGGT TTACTGGTTA TTCTTTCATATCGATGTGGG AATCAAT GAACACC-3′ TCC-3′ Bordetella baf Strain TP177 TP178pertussis ATCC 9797 5′-GGGTCTA 5′-GGGATCC genomic DNA GAAAAGGAGGTTAGGCCGTT AATTTAAATG GGCGCGCCTT ATTATCCTCA GCGCGG TCGACTCCG CG-3′ GC-3′Borrelia BB0527 Strain TP171 TP172 burgdorferi ATCC 35210 5′-GGGTCTA5′-GGGATCC genomic DNA GAAAAGGAGG TTAATTAACA AATTTAAATG AACTTAAAGTAATAAACCTT CAATAGAATT TATTATCAGA TCCTAAAATT ATTGATAATT CTAACGCCTTGATATTGGAA CTACAG-3′ ATACCA GC-3′ Helicobacter HP0862 Strain TP167 TP168pylori 26695 ATCC 700392 5′-GGGTCTA 5′-GGGATCC genomic DNA GAAAAGGAGGTTATTTGCAT AATTTAAATG TCTAGTATCC CCAGCTAGGC CTGCTTTTTT AATCTTTTACAAGAGCGATT AGATTTGAAA TCCATCCC AACCTGG-3′ GTC-3′ Pseudomonas PA4279Strain TP169 TP170 aeruginosa ATCC 47085 5′-GGGTCTA 5′-GGGATCC PA01genomic DNA GAAAAGGAGG TTACTCAATC AATTTAAATG GGGCAAGCCA ATTCTTGAGCGTGCCAGCCC TCGACTGTGG TACG-3′ AAACTCG CTG-3′

The purified PCR products were cloned by ligation with plasmid vectorpASK-1BA3 (Sigma-Genosys) which had been digested with XbaI and BamHIfollowed by transformation into strains LH-1 and XL1-Blue/MRF'kan.Plasmids containing inserts were identified by restriction enzymedigestion of plasmid DNA isolated from selected transformants. Examplesof plasmids containing the H. pylori (pOTP72), P. aeruginosa (pOTP73),or B. subtilis (pOTP71) coaX gene are shown in FIGS. 8, 9 and 10,respectively. The identity of inserts in plasmids is confirmed by DNAsequence analysis.

The pantothenate kinase activity of each of the above cloned coaXhomologs can be demonstrated by transforming the plasmids describedabove into E. coli strain YH1 containing the coaA15(Ts) mutation andlooking for complementation at the non-permissive temperature of 43°-44°C. For example, as shown in Table 4, transformation of E. coli YH1containing the coaA15(Ts) with plasmid pOTP72 containing the cloned H.pylori coaX gene (HP0862) or plasmid pOTP73 containing the cloned P.aeruginosa coaX gene (PA4279) enabled the E. coli cells with thetemperature sensitive coaA gene product to grow at 44° C. as is also thecase when these cells were transformed with the plasmid containing theB. subtilis coaX gene (pOTP71). These experiments confirm that the coaXhomologs in H. pylori and P. aeruginosa due indeed each encode an activepantothenate kinase.

TABLE 4 Transformation of YH1 (coaA15(Ts)) with coaX ligation mixturesand control plasmid DNA Number of colonies Number of colonies DNA at 30°C. at 44° C. NONE zero zero Ligated, cut vector 5 zero Uncut vector >500zero (pASK-1BA3) B. subtilis coaX, 74 67 pool A ligation B. subtiliscoaX, 230 160 pool B ligation H. pylori coaX 53 38 (HP0862) pool Aligation H. pylori coaX 99 56 (HP0862) pool B ligation P. aeruginosacoaX 366 279 (PA4279) pool A ligation P. aeruginosa coaX 282 359(PA4279) pool B ligation

Since the coaX homologs cloned in pASK-1BA3 were inserted downstream ofa Tet-inducible promoter, enzyme for in vitro screening assays can beobtained by inducing gene expression as described by Sigma-Genosys, andthen isolating the overproduced pantothenate kinase by conventionalprotein purification procedure. Alternatively, the coaX gene can becloned into any of various protein or peptide fusion expression vectorsthat facilitate purification of the protein. For example, Helicobacterpylori, Borrelia burgdorferi, Bordetella pertussis, and Pseudomonasaeruginosa coaX genes can be cloned into protein fusion expressionvectors such as those available from companies including but not limitedto Qiagen™ or Invitrogen™ to produce a His tagged CoaX fusion proteinsor glutathione-S-transferase/CoaX fusion proteins which can be isolatedby binding to nickel affinity or glutathione sepharose resins,respectively.

Equivalents Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

1. A method for identifying compounds that inhibit pantothenate kinaseactivity, comprising: a) contacting a cell comprising a recombinantexpression vector expressing a polypeptide with pantothenate kinaseactivity with a test compound, wherein said polypeptide is encoded by acoaX gene; and b) determining the ability of the test compound toinhibit pantothenate kinase activity in said cell; wherein inhibition ofpantothenate kinase activity identifies the compound as an inhibitor ofpantothenate kinase activity.
 2. The method of claim 1, wherein therecombinant cell is dependent on the polypeptide encoded by the coaXgene for growth.
 3. The method of claim 1, wherein the recombinant cellis not dependent on the polypeptide encoded by the coaX for growth. 4.The method of claim 1, wherein the recombinant cell further comprises acoaA gene.
 5. The method of claim 4, wherein the coaA gene is expressedby a recombinant expression vector.
 6. A method for identifyingcompounds that inhibit CoaX pantothenate kinase activity, comprising: a)contacting a cell comprising a recombinant expression vector expressinga polypeptide encoded by a coaX gene with a test compound, wherein saidcell is dependent on the polypeptide encoded by the coaX gene forgrowth; b) determining the ability of the test compound to inhibitgrowth of said cell; and c) comparing the ability of the cell comprisingthe recombinant expression vector expressing the polypeptide encoded bythe coaX gene to grow with the ability of a cell comprising a coaA geneto grow; wherein the inability of the cell comprising the recombinantexpression vector expressing the polypeptide encoded by the coaX gene togrow and the ability of the cell comprising the coaA gene to growidentifies the compound as an inhibitor of CoaX pantothenate kinaseactivity.
 7. The method of claim 4 or 6, wherein the cell comprising thecoaA gene encodes a pantothenate kinase polypeptide with reducedactivity.
 8. The method of claim 7, wherein said reduced pantothenatekinase activity is due to an environmental manipulation.
 9. The methodof claim 6 or 7, wherein the coaA gene is expressed by a recombinantexpression vector.
 10. The method of claim 1 or 6, wherein said cellcomprising the recombinant expression vector expressing a polypeptideencoded by a coaX gene is a Gram negative microorganism.
 11. The methodof claim 1 or 6, wherein said cell comprising the recombinant expressionvector expressing a polypeptide encoded by a coaX gene is a Grampositive microorganism.
 12. The method of claim 6, wherein said cellcomprising the coaA gene is a Gram negative microorganism.
 13. Themethod of claim 6, wherein said cell comprising the coaA gene is a Grampositive microorganism.
 14. The method of claim 1 or 6, wherein saidcell comprising the recombinant expression vector expressing apolypeptide encoded by a coaX gene is of the genus Bacillus orEscherchia.
 15. The method of claim 1 or 6, wherein said cell comprisingthe recombinant expression vector expressing a polypeptide encoded by acoaX gene is Bacillus subtilis or Escherchia coli.
 16. The method ofclaim 6, wherein said cell comprising the coaA gene is of the genusBacillus or Escherchia.
 17. The method of claim 6, wherein said cellcomprising the coaA gene is Bacillus subtilis or Escherchia coli. 18.The method of claim 1 or 6, wherein said cell comprising the recombinantexpression vector expressing a polypeptide encoded by a coaX gene is anon-pathogenic bacterium and said coaX gene is derived from a pathogenicbacterium.
 19. The method of claim 1 or 6, wherein said cell comprisingthe recombinant expression vector expressing a polypeptide encoded by acoaX gene is selected from the group consisting of PA861, PA876, YH1comprising pOTP71, YH1 comprising pOTP72, YH1 comprising pOTP73, and YH1comprising pAN341.